*Ecological Archives* E093-107-A3

Sarah A. Orlofske, Robert C. Jadin, Daniel L. Preston, and Pieter T. J. Johnson. 2012. Parasite transmission in complex communities: Predators and alternative hosts alter pathogenic infections in amphibians. *Ecology* 93:1247–1253.

Appendix C. Methods and results of alternative prey bioassays conducted in the laboratory with damselfly and fish predators.

*Methods*

We conducted additional bioassay trials to assess the ability of mosquitofish (*Gambusia affinis*) or damselfly nymphs (*Enallagma* spp.) to consume *Ribeiroia ondatraee* cercariae in the presence of alternative prey. Each predator was fasted for 24 hr. prior to assay to standardize hunger and all trials were conducted in the morning (0200–0600hr) corresponding to peak cercarial shedding. We had three treatments: 20 *R. ondatrae* cercariae and 10 *Daphnia middendorffiana*, 15 of each taxa, and 10 *R. ondatrae* cercariae with 20 * D. middendorffiana*. Each treatment was replicated five times for each species. Each individual was placed into 60 ml of water for 30 minutes of acclimation before the prey were added. After 30 minutes, predators were removed and the remaining prey of each taxa were immediately counted using a dissecting microscope. We included replicates of each treatment with no predator present to ensure we could recover all prey items offered as a control treatment to assess parasite recovery.

To determine whether predators showed a prey preference for *Daphnia* or cercariae, we calculated the 'case 2' form of Chesson's α, which is a prey selectivity index that is appropriate for situations when prey is removed without replacement (Chesson 1978, Chesson 1983). Chesson's α was calculated using the index:

where *n*_{i0} is the number of individuals of prey type *i* present at the beginning of the trial, *r*_{i} is the number of individuals of prey type *i* remaining at the end of the trial and *m* is the number of prey types. Values for α_{i} can range between zero (perfect prey avoidance) and one (perfect prey selection). We used a one-sample *t*-test to compare the mean Chesson's α value for cercariae to the expected value of 1/*m* (i.e, 0.5 with two prey types), which is the value of Chesson's α that is expected with random prey selection.

*Results*

Fewer than 27% of the *Daphnia* and 33% of the cercariae were consumed in any one of the damselfly predation trials. Chesson's α for cercariae consumed by damselflies was 0.57 (SE = 0.09), which was not significantly different from the null value of 0.5 under random prey selection (*P* = 0.44, *t* = 0.79, df = 16). This result suggests that damselflies are likely to prey on cercariae when alternative prey such as *Daphnia* are available. Mosquitofish in the alternative prey trials with *Daphnia* and cercariae consumed all of the available prey in 14 out of the 17 successful trials. Of the trials where prey items were not completely depleted, there was one trial with a single remaining *Daphnia*, one trial with two remaining cercariae, and one trial with one *Daphnia* and five remaining cercariae. Because the prey were depleted in most of the trials, we did not use Chesson's α to calculate prey selectivity for the mosquitofish. While these results do not allow us to determine whether cercariae or *Daphnia* are preferred prey, it nonetheless indicates that cercariae are still consumed when other prey are available simultaneously and the mosquitofish have not been fasted prior to being offered cercariae.

Literature Cited

Chesson, J. 1978. Measuring preference in selective predation. Ecology 59:211–215.

Chesson, J. 1983. The estimation and analysis of preference and its relationship to foraging models. Ecology 64:1297–1304.